Method of synthesizing a suppressor tRNA, DNA construct and use thereof for producing a non-natural amino acid-incorporated protein

ABSTRACT

There are provided a DNA construct comprising a suppressor tRNA gene of a non-eukaryote containing no internal promoter functioning in a eukaryotic cell, and a eukaryotic or bacteriophage promoter linked at the 5′ end of the tRNA gene, a method for synthesizing a suppressor tRNA by using the DNA construct, and a process for producing protein incorporating a non-natural amino acid by using the same.

RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser.No. 12/196,129 filed on Aug. 21, 2008, which is a ContinuationApplication under 35 USC §120 of International Application No.PCT/JP2007/05334, filed on Feb. 22, 2007, which in turn claims priorityunder 35 USC §119 of Japanese Application No. 2006-045788, filed on Feb.22, 2006.

TECHNICAL FIELD

The present invention relates to a method of synthesizing a tRNA and aDNA construct therefor, particularly to a method of synthesizing asuppressor tRNA corresponding to a non-natural amino acid and a DNAconstruct therefor, as well as a method of producing a proteinincorporating a non-natural amino acid using the above.

BACKGROUND ART

A protein incorporating a non-natural amino acid (hereinafter alsoreferred to as an “alloprotein”) in which an amino acid residue at adesired position in a protein is replaced with an amino acid other than20 different amino acids involved in normal protein synthesis (anon-natural amino acid) could offer an effective means of analyzing thefunction and structure of a protein. Meanwhile, lysine derivativesinclude amino acids, such as acetyl-lysine, methyl-lysine etc., whichare synthesized by post-translational modification. Such amino acids arewell-known particularly as those involved in regulation of geneexpression by histones and are also known as those involved inregulation of transcriptional activation, regulation of protein-proteininteraction, and suppression/promotion of ubiquitination for many typesof proteins. It is expected that many findings concerning acetylation,methylation etc. of lysine could be made if those lysine derivativescould be introduced site-specifically into a protein synthesized by aeukaryote.

Pyrrolysyl tRNA synthetase (PylRS) is a novel aminoacyl tRNA synthetase(aaRS) found in a methanogenic archaebacterium (Methanosarcina). Acorresponding tRNA (pyrrolysine tRNA) is a suppressor tRNA, which has aunique secondary structure such as an unusually small D loop, etc.Recently, it was found that in Escherichia coli, PylRS and pyrrolysinetRNA do not interact with endogenous aaRS and tRNA (orthogonality), andpyrrolysine could be introduced specifically into the site of an ambercodon in a protein (Non-Patent Document 1). Further, it has beenreported that a wild-type PylRS can bind a non-natural amino acid suchas Nε-Boc-L-lysine to pyrrolysine tRNA in Escherichia coli (Non-PatentDocument 1).

On the other hand, in a mammalian cell, enzymes that phosphorylatetyrosine residues in proteins (tyrosine kinases) play an important rolein transducing extracellular signals, such as by growth stimulatingfactors, into the nucleus. The tyrosine kinases include one capable ofphosphorylating a tyrosine derivative and one incapable ofphosphorylating a tyrosine derivative. For example, it was shown that aSrc kinase phosphorylates an iodotyrosine residue but an EGF receptorcannot do so. Thus, it is useful in examining interaction of a desiredprotein with various tyrosine kinases in a cell if an alloprotein, thedesired protein into which a tyrosine derivative is incorporated, couldbe synthesized in a mammalian cell. For example, it is important inanalysis of signal transduction mechanisms to examine which tyrosinekinase phosphorylates the desired protein. Further, these non-naturalamino acid-incorporated proteins could be useful in themselves asmaterial for analysis of the function and structure of a protein, andcould have a novel bioactivity.

As an expression method of an alloprotein like the above in an animalcell, there has been developed a method of expressing in an animal cell(A) a mutant tyrosyl tRNA synthetase (hereinafter referred to as “mutantTyrRS”), which is a variant of a tyrosyl tRNA synthetase derived fromEscherichia coli and has an increased specificity to a non-naturaltyrosine derivative as compared with the specificity to a tyrosine, (B)a suppressor tRNA originating from eubacterium, such as bacillus,mycoplasma, and staphylococcus, and capable of binding to the abovetyrosine derivative in the presencGe of the above mutant tyrosyl tRNAsynthetase, and (C) a desired protein gene including a nonsense mutationor frame shift mutation at a desired position. The above tyrosinederivative has been incorporated into the position of the nonsensemutation or frame shift mutation of the above protein (Patent Document 1and Non-Patent Document 2).

Hereupon, it is required that the above suppressor tRNA originating fromthe non-eukaryote is transcribed by an RNA polymerase in a eukaryoticcell. In contrast to one kind of RNA polymerase in prokaryotic cells, itis known that in eukaryotic cells, there are three different kind of RNApolymerases I, II, and III (polI, polII, and polIII) that sharefunctions. PolI synthesizes ribosomal RNA, PolII synthesizes mRNA, andPolIII synthesizes 5S rRNA, tRNA, U6 small nuclear RNA (snRNA), etc.Therefore, tRNA in a eukaryotic cell is synthesized by transcription byRNA polymerase III. Genes transcribed by the RNA polymerase III areclassified broadly into three groups according to characteristics oftheir promoter structures, the groups including, as their representativegenes, a 5S rRNA gene (Type I promoter), a tRNA gene (Type II promoter),and a U6 small nuclear RNA gene (Type III promoter), respectively. Thetype II promoter, which transcribes a tRNA, is an internal promoter madeup of two regions in a tRNA coding sequence, the consensus sequences ofwhich are known as box A and box B. The consensus sequence of the box Aconsists of the positions 8-19: TRGCNNAGYNGG (SED ID NO:1), and theconsensus sequence of the box B consists of the positions 52-62:GGTTCGANTCC (SED ID NO:2). Accordingly, for example, the suppressortyrosine tRNA of Bacillus stearothermophilus, although it originatesfrom a prokaryote, can be expressed in an animal cell without anyalterations, because of the presence of the box A and box B in itssuppressor tyrosine tRNA coding sequence (refer to Non-patent Document3, for example).

Here, incorporation of an amino acid into the position of the nonsensemutation in the above protein is referred to as suppression. Becausethere are only three different types of stop codons, a maximum of threetypes of non-natural amino acids can be incorporated into one type ofprotein. In vitro experiments have developed artificial base pairs inaddition to naturally occurring base pairs (refer to Non-patentDocuments 4 and 5), and an RNA containing artificial base pairs asmentioned above can be transcribed in vitro by using an RNA polymeraseof T7 bacteriophage. It is expected that the following could beachieved: increase in the number of codon types, which are now 4³, byusing artificial base pairs in codons encoding amino acids, andintroduction of a plurality of non-natural amino acids into one type ofprotein by getting the codons that do not encode natural amino acids toencode non-natural amino acids.

-   [Patent Document 1] WO2004/039989A1-   [Non-Patent Document 1] Blight, S. K. et al., Nature, 431, 333-335    (2004)-   [Non-Patent Document 2] Sakamoto, K. et al., Nucleic Acids Research    30, 4692-4699 (2002)-   [Non-Patent Document 3] M. Sprinzl et al., Nucleic Acids Research    17, 1-172 (1989)-   [Non-Patent Document 4] Hirao, I. et al., Nature Biotechnology 20,    177-182 (2002)-   [Non-Patent Document 5] Hirao, I. et al., Nature Methods 3, 729-735    (2006)

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

In the case of the above-mentioned expression of a suppressor tRNAoriginating from a non-eukaryote in a eukaryotic cell, however, theproblem is raised that, if the tRNA has sequences significantlydifferent from the consensus sequences of the eukaryote in place of boxA and box B, the sequences do not function as an internal promoter, andonly an extremely small amount of transcription is achieved in aneukaryotic cell or almost no transcription occurs therein. For example,the D loop of a pyrrolysine tRNA originating from methanogenicarchaebacterium, which lacks several bases and is unusually small, doesnot function as internal promoter in a eukaryotic cell. Further, asuppressor tyrosine tRNA of Escherichia coli has the box B consensussequence in its sequence but does not contain the box A consensussequence. Introduction of the boxes A and B into those tRNAs results inloss of their functions as tRNA so that alloproteins with anincorporated lysine derivative or tyrosine derivative cannot besynthesized even if a pyrrolysine tRNA or an E. coli suppressor tyrosinetRNA including boxes A and B is used.

On the other hand, it is unknown whether or not those suppressor tRNAshaving no internal promoter would function as tRNA in cases where thesuppressor tRNAs are expressed using an external promoter in aeukaryotic cell. That is, although it is required that base modificationand formation of 3-dimentional structure, etc. after transcriptionnormally occur in order that a tRNA functions, it remains unknown wherea tRNA transcribed by an external promoter other than a type II promoterwould be localized in the cell, whether it would undergopost-transcriptional modification or not, and further whether it wouldpresent biological functions or not.

Means to Solve the Problems

After investigations and considerations, the inventors have found outthat a pyrrolysine tRNA originating from methanogenic archaebacterium ora suppressor tyrosine tRNA of Escherichia coli can be efficientlyexpressed in an animal cell by binding to its 5′ end a promoter sequenceof a eukaryotic tRNA nucleotide sequence or U1 and U6 snRNA gene(s).Further, it has been found out that the tRNA can be efficientlyexpressed by binding a bacteriophage-originating promoter sequence tothe 5′ end of the tRNA gene and introducing the promoter together with aRNA polymerase capable of transcription into an animal cell. The presentinvention has been accomplished based on those findings.

In accord with a first aspect of the present invention, there isprovided a DNA construct comprising a suppressor tRNA gene of anon-eukaryote and lacking an internal promoter sequence that functionsin a eukaryotic cell, and a promoter of a eukaryote linked to the 5′ endof the tRNA gene. It is preferred that the tRNA gene is a pyrrolysinetRNA gene originating from an archaebacteria and/or a suppressortyrosine tRNA gene originating from Escherichia coli, and the DNAconstruct further comprises a transcription terminator sequence linkedto the 3′ end of said tRNA gene. In a further preferable exemplaryembodiment, the promoter of a eukaryote is a nucleotide sequence thatinduces transcription by RNA polymerase II or III. The nucleotidesequence that induces the transcription by the RNA polymerase II ispreferably a promoter of a U1 snRNA gene, for example. Also, it isparticularly preferred that the nucleotide sequence that induces thetranscription by the RNA polymerase III is a promoter of a eukaryotictRNA gene such as a human valine tRNA nucleotide sequence, or a promoterof a U6 snRNA gene, for example.

In accord with a second aspect of the present invention, there isprovided i) a method of synthesizing a suppressor tRNA comprisingcausingthe DNA construct to undergo transcription in a eukaryotic cell; ii) arecombinant eukaryotic cell that is transformed or transfected by theDNA construct; and iii) a method of synthesizing an aminoacyl-tRNA,comprising expressing a tRNA transcribed by the DNA construct and anaminoacyl-tRNA synthetase corresponding to said tRNA.

In accord with a third aspect of the present invention, there isprovided a process for producing a protein incorporating a non-naturalamino acid comprising: expressing, in the presence of the non-naturalamino acid in a eukaryotic cell, (a) an aminoacy-tRNA synthetase for thenon-natural amino acid, (b) a tRNA which is capable of binding to thenon-natural amino acid in the presence of the aminoacyl-tRNA synthetase,and which is transcribed from the DNA construct, and (c) a desiredprotein that has a nonsense mutation or frame shift mutation at adesired position.

In accord with a forth aspect of the present invention, there isprovided a DNA construct comprising: a suppressor tRNA gene from anon-eukaryote lacking an internal promoter sequence that functions in aeukaryotic cell, and a promoter originating from a bacteriophage linkedto the 5′ end of the tRNA gene. It is preferred that the tRNA gene is apyrrolysine tRNA gene originating from archaebacteria and/or asuppressor tyrosine tRNA gene originating from Escherichia coli. The DNAconstruct further comprises a transcription terminator sequence linkedto the 3′ end of the tRNA gene. The bacteriophage promoter ispreferably, but not restricted to, a T7 promoter, T3 promoter, or SP6promoter.

In accord with a fifth aspect of the present invention, there areprovided a method of synthesizing a suppressor tRNA comprising causing aDNA construct to undergo transcription in a eukaryotic cell, the DNAconstruct comprising a suppressor tRNA gene of a non-eukaryote lackingan internal promoter sequence that functions in a eukaryotic cell, and apromoter originating from bacteriophage linked to the 5′ end of saidtRNA gene; and a recombinant eukaryotic cell being transformed ortransfected by the DNA construct and a gene expressing an RNA polymerasecorresponding to the bacteriophage promoter.

In accord with a sixth aspect of the present invention, there isprovided a process for producing a protein including a non-natural aminoacid comprising i) preparing a DNA construct comprising a suppressortRNA gene of a non-eukaryote and lacking an internal promoter sequencethat functions in a eukaryotic cell, and a promoter from a bacteriophageoperably linked to the 5′ terminal region of the tRNA gene, wherein thesuppressor tRNA is capable of binding to the non-natural amino acid inthe presence of an aminoacyl-tRNA synthetase for the non-natural aminoacid; and ii) expressing, in the presence of the non-natural amino acidin a eukaryotic cell, (a) a tRNA transcribed from the DNA construct, (b)an aminoacyl-tRNA synthetase for the non-natural amino acid, and (c) adesired protein that has a nonsense mutation or frame shift mutation ata desired position. The tRNA gene is preferably, but not restricted to,a pyrrolysine tRNA gene originating from archaebacteria and/or asuppressor tyrosine tRNA gene originating from Escherichia coli.Further, it is preferable that the non-natural amino acid is, but notrestricted to, a lysine derivative or a tyrosine derivative.

In accord with a seventh aspect of the present invention, there isprovided a process for producing a protein including a non-natural aminoacid comprising: preparing a DNA construct comprising a suppressor tRNAgene of a non-eukaryote lacking an internal promoter sequence thatfunctions in a eukaryotic cell, and a bacteriophage promoter operablylinked to the 5′ terminal region of the tRNA gene, wherein thesuppressor tRNA is capable of binding to the non-natural amino acid inthe presence of an aminoacyl-tRNA synthetase for the non-natural aminoacid; and expressing, in the presence of the DNA construct and thenon-natural amino acid in a eukaryotic cell, (a) an RNA polymerasecorresponding to the bacteriophage promoter, (b) an aminoacyl-tRNAsynthetase for the non-natural amino acid, and (c) a desired proteinthat has a nonsense mutation or frame shift mutation at a desiredposition. The tRNA gene is preferably, but not restricted to, apyrrolysine tRNA gene originating from archaebacteria and/or asuppressor tyrosine tRNA gene originating from Escherichia coli.Further, it is preferable that the non-natural amino acid is, but notrestricted to, a lysine derivative or a tyrosine derivative

Meritorious Effects of the Invention

Using a process in the present invention allows a tRNA originating froma non-eukaryote and an aminoacyl-tRNA, to be efficiently expressed in aeukaryotic cell, and a non-eukaryotic suppressor tRNA containing nointernal promoter sequences (box A, box B) that function in a eukaryoticcell to be expressed in a eukaryotic cell. It is expected that using theprocess of the present invention allows expression of aminoacyl-tRNAcontaining an artificial, non-natural base in a eukaryotic cell, andsynthesis of an alloprotein containing 4 or more different types ofnon-natural amino acids.

Further, using a process in the present invention allows synthesis of analloprotein, particularly in eukaryotes, into which there isincorporated a lysine derivative such as Nε-acetyl-lysine,Nε-trimethyl-lysine, Nε-t-butoxycarbonyl-lysine,Nε-2-methylamino-benzoyl-lysine containing a fluorescent group, etc., byusing a wild-type aminoacyl-tRNA synthetase originating fromarchaebacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a cloverleaf structure of a pyrrolysine tRNA.

FIG. 2 shows a result of suppression of Grb2 (111 amb) detected bywestern blot in Example 1.

FIG. 3 shows a result of suppression of Grb2 (111 amb) detected bywestern blot in Example 2.

FIG. 4 shows a result of lacZ amber suppression in the case oftRNA^(Pyl) being expressed by using 3 different promoters or enhancers.

FIG. 5 shows a result of lacZ amber suppression by tRNA^(Tyr) linked toa U6 promoter in cases where 3 different non-natural amino acids wereadded.

FIG. 6 shows mass spectrum data demonstrating that in Escherichia coli,Nε-Boc-lysine was incorporated into a peptide in the presence of PylRSand pyrrolysine tRNA.

FIG. 7 shows a result of suppression of lacZ (91 amber) in the case oftRNA^(Pyl) being expressed by using a T7 promoter in Example 5. It isapparent therefrom that in the case of T7 RNA polymerase being expressed(T7RNAP+), β-galactosidase activity detected is significantly high ascompared with the case when T7 RNA polymerase is not expressed(T7RNAP−), and the amber codon of a lacZ gene is suppressed.

FIG. 8 shows a result of suppression of lacZ (91 amber) in the case oftRNA^(Tyr) being expressed by using T7 promoter in Example 5. It isapparent therefrom that in the case of T7 RNA polymerase being expressed(T7RNAP+), β-galactosidase activity detected is significantly high ascompared with the case when T7 RNA polymerase is not expressed(T7RNAP−), and the amber codon of a lacZ gene is suppressed.

FIG. 9 shows a result of suppression of lacZ (91 amber) detected bycellular staining in the case of tRNA^(Tyl) being expressed by using aU1 snRNA transcription promoter in Example 6.

PREFERRED MODES FOR CARRYING OUT THE INVENTION Non-Natural Amino Acid

A non-natural (non-naturally occurring) amino acid as may be used hereinincludes, for example, a lysine derivative or a tyrosine derivative. Asa lysine derivative, a non-natural amino acid is preferably one in whichthe hydrogen atom bonded to the nitrogen atom at the ε position isreplaced with another atom or chemical group. Lysine derivatives includepyrrolysine, Nε-t-butoxycarbonyl-lysine (Nε-Boc-lysine),Nε-acetyl-lysine, Nε-trimethyl-lysine, andNε-2-methylamino-benzoyl-lysine (Nma-lysine), for example. Site-specificincorporation of methyllysine or acetyllysine, which are modifiedlysines present in a eukaryote, into a protein could produce manyfindings regarding methylation or acetylation of lysine. Suchalloprotein with the lysine derivative incorporated is useful asmaterial for analysis of function and structure of the protein, andcould offer a target for drug development. Tyrosine derivatives include3- or 4-substituted tyrosine made up of a tyrosine having a substituentat 3- or 4-position of the phenyl group thereof. 3-substituted tyrosineincludes 3-halogenated tyrosine such as 3-iodotyrosine and3-bromotyrosine. 4-substituted tyrosine includes4-acetyl-L-phenylalanine, 4-benzoyl-L-phenylalanine,4-azido-L-phenylalanine, O-methyl-L-tyrosine, 4-iodo-L-phenylalanineetc. Those amino acids can be prepared by known methods and arecommercially available.

(Aminoacyl-tRNA Synthetase)

Aminoacyl-tRNA synthetase as used herein is tRNA synthetase capable ofrecognizing a non-natural amino acid and specifically recognizing asuppressor tRNA to produce a suppressor tRNA connected to suchnon-natural amino acid.

In a preferred exemplary embodiment, PylRS originating from amethanogenic archaebacterium is provided that is able to recognize, asan amino acid a lysine derivative and specifically recognize, as tRNA, apyrrolysine tRNA (SEQ ID NO:4) used in combination to produce asuppressor tRNA connected to such lysine derivative. A methanogenicarchaebacterium is preferably Methanosarcina mazei (M. mazei). PylRS isexpressed in a eukaryotic cell, preferably in an animal cell,particularly preferably in a mammalian cell. In order to express PylRSin a cell, for example, a plasmid may be introduced into the mammaliancell which plasmid is constructed such that a DNA sequence made up of awild-type gene originating in Methanosarcina mazei, added with FLAG tag,etc., at its N terminal region, is amplified using PCR, followed byincorporation of the resultant DNA sequence into the NheI-BamHI site ofcommercially available pcDNA3.1 (Invitrogen), p. 107 (Cytotechnology, 3,133 (1990)), p. 103 [J. Biochem. 101, 1307 (1987)], etc.

In other exemplary embodiments, there can be used various variants ofTyrRS originating in Escherichia coli and capable of specificallyrecognizing a tyrosine derivative to produce a suppressor tRNA (SEQ IDNO:5) connected with the tyrosine derivative. For example, the E. coliTyrRS variant (V37C195) specifically recognizes 3-iodotyrosine.Alternatively, it has been reported that a TyrRS variant made up of E.coli TyrRS with five amino acid mutations introduced at positions 37,126, 182, 185 and 186 recognized non-natural amino acids such as4-azido-L-phenylalanine and 4-benzoyl-L-phenylalanine, etc. (Chin, J. W.Et al., Science, 301, 964-967, 2003). Wild-type E. coli TyrRS does notreact with tRNA^(Tyr) of eukaryotes, and tRNA^(Tyr) of prokaryotes doesnot react with TyrRS of eukaryotes.

(tRNA)

It is required for tRNA used in combination with the aboveaminoacyl-tRNA synthetase to satisfy the requirements 1) that it isassigned to a nonsense codon other than codons assigned to usual 20different amino acids, 2) that it is recognized only by the abovenon-natural amino acid-specific aminoacyl-tRNA synthetase but notrecognized by an aminoacyl-tRNA synthetase normally present in a hostcell (orthogonal tRNA) and 3) that is can be expressed in a eukaryoticcell. In a case where the aminoacyl-tRNA synthetase is PylRS, thecorresponding pyrrolysine tRNA is a tRNA originating in a non-eukaryoticcell that has an anti-codon complementary to a nonsense codon and a3-dimensional structure for functioning as suppressor tRNA, and it isexpressed in a eukaryotic cell. That is, in this case, the tRNA is asuppressor tRNA that satisfies the requirement that it is assigned to anonsense codon other than codons assigned to usual 20 different aminoacids, and recognized only by the above lysine derivative-specific PylRSbut not recognized by an aaRS normally present in a host cell(orthogonality); and is expressed in an animal cell.

Here, nonsense codons include UAG (amber), UAA (ochre), UGA (opal) etc.,but UAG (amber) is preferably used. Instead of a nonsense codon, a codonmade up of 4 or more bases (preferably 4 or 5 bases) (hereinafterreferred to as a “frame shift codon”) may be used.

As mentioned above, expression of tRNA in a eukaryotic cell requires twointernal promoters in a tRNA coding sequence, the consensus sequences ofwhich are known as box A and box B. FIG. 1 shows a cloverleaf structureof a pyrrolysine tRNA. In FIG. 1, the mark ◯ in the loop at the left (Dloop) indicates lack of a base. As shown in FIG. 1, the pyrrolysine tRNAlacks 3 bases in the D loop and is extraordinarily small as comparedwith the D loops of the other tRNAs. In order to express the pyrrolysinetRNA in an animal cell, the box A and B sequences were incorporated intothe pyrrolysine tRNA, but this resulted in a drastic change in thestructure of the tRNA because of the anomalously small size of the Dloop, and in a failure in retaining its suppressor activity.

(Synthesis of tRNA, aminoacyl-tRNA)

In a method of synthesizing aminoacyl-tRNA according to the presentinvention, a tRNA of a non-eukaryote containing no internal promotersequence that functions in a eukaryotic cell, a eukaryote-originatingpromoter being linked to the 5′ end of the tRNA, is caused to undergotranscription in a eukaryotic cell, preferably in an animal cell,particularly preferably in a mammalian cell, which contains anaminoacyl-tRNA synthetase. In this case, it is preferable that atranscription terminator sequence is linked to the 3′ end of the tRNA.To be more specific, an aminoacyl-tRNA of the present invention isobtained in the following manner: the sequence of a wild-typepyrrolysine tRNA of Methanosarcina mazei was synthesized from DNAprimers, the 5′ end of which is linked to a eukaryotic promoter and the3′ end of which is linked to a transcription terminator sequence, andthis construct is incorporated into, for example, pcDNA3.1 orpCR4Blunt-TOPO (both available from Invitrogen). The resulting plasmidis introduced into an animal cell to express the tRNA, followed bytranscription and processing in the animal cell.

As the above eukaryote-derived promoter, there can be used a nucleotidesequence that induces transcription by an RNA polymerase II or III. Thenucleotide sequence that induces transcription by an RNA polymerase IIis preferably a U1 snRNA gene promoter. However, it has been reportedthat a U6 snRNA gene promoter with mutated TATA box region acts aspromoter of the U1 snRNA gene and thus such promoter may be used. Thepromoter nucleotide sequence that induces transcription by an RNApolymerase III is preferably a eukaryotic tRNA gene or U6 snRNA genepromoter. In this case, it is preferred that the eukaryotic tRNA gene islinked via a linker to the 5′ end of a wild-type pyrrolysine tRNA gene.The linker includes, but is not restricted to, a linker cleaved byBglII, XbaI, XhoI etc. The tRNA gene linked to the 5′ end is oneoriginating from a eukaryote, which includes, but is not restricted to,an animal, a plant, an insect, etc. Among these, a human tRNA gene ispreferable. An amino acid to which the tRNA should be linked may be anyone of the usual 20 different naturally-occurring amino acids,preferably valine among them.

Human U6 small nuclear RNA (snRNA) is an RNA species which is abundantlypresent in a spliceosome that is formed at the stage of splicing bypre-mRNA and reaches 4−5×10⁵ copies per cell. The U6 promoter isbelieved to drive transcription of a small heterologous RNA, theactivity of the transcription being higher than the activity oftranscription using a tRNA promoter. Although the U6 promoter and thetRNA promoter are both transcribed by PolIII, both are different fromeach other in location: i.e., the U6 promoter is located at 5′ upstreamof the structural gene whereas the tRNA promoter is located in theinterior of its own structural gene. The human U6 snRNA promoter hasdistinctive promoter elements known as an enhancer region (or distalpromoter region) and a core region (or proximal promoter region), and ispreferably a nucleotide sequence, which is formed of the nucleotidesequence set forth in SEQ ID NO:3 or a nucleotide sequence being atleast 30%, 50%, 70%, 80%, 90% or 95% homologous to the nucleotidesequence set forth in SEQ ID NO:3, and which has the activity oftranscription by an RNA polymerase III in a mammalian cell. The degreeof homology between nucleotide sequences can be represented bypercentage of identity of two appropriately aligned nucleotidesequences, which means incidence of accurately identical amino acidsbetween the sequences. Appropriate alignment between sequences foridentity comparison may be determined using, for example, the BLASTalgorithm (Altschul SF J Mol Biol 1990 Oct. 5; 215(3):403-10).

In the present invention the above tRNA can be efficiently transcribedin a eukaryotic cell also by using a promoter originating frombacteriophage. To be specific, possible bacteriophage promoters include,but are not restricted to, a T7 promoter, T3 promoter and SP6 promoteroriginating from bacteriophage of Escherichia coli. These promoters maybe inserted into any positions within the 5′ terminal region of theabove tRNA gene, but the insertion position is preferably 10-50 bpupstream from the transcription initiation site of the gene. In the caseof a bacteriophage promoter being used, it is required to use aeukaryotic cell in which a bacteriophage RNA polymerase corresponding tosuch promoter is expressed. To be specific, T7 RNA polymerase, T3 RNApolymerase, and SP6 RNA polymerase may be used with the above promoters.It has been reported that when expressed in a mammalian cell, T7 RNApolymerase caused transcription of RNA from DNA containing a T7 promotersequence, and the amount of the transcribed RNA reached a maximum of 20%of the total RNAs in the cell. It has been reported that, as in the caseof a T7 RNA polymerase, a T3 RNA polymerase and a SP6 RNA polymerasehave been used, the promoters thereof being as short as 20 bp or lesslike the T7 promoter, and the RNA polymerases each having substantiallythe same ability of RNA transcription (as the T7 RNA polymerase) toprepare a large amount of RNA in a mammalian cell. The T7 promoter ofthe nucleotide sequence set forth in SEQ ID NO:13, or a sequence formedof a nucleotide sequence being at least 70%, 80%, 90% or 95% homologousto the nucleotide sequence set forth in SEQ ID NO:13 and being capableof inducing transcription by a T7 RNA polymerase in a mammalian cell ispreferred as a bacteriophage promoter.

(Protein into which a Non-Natural Amino Acid is Incorporated)

In the present invention, proteins into which a non-natural amino acidis incorporated are not restricted to particular types. Such proteinsmay be any proteins capable of expression and further can beheterologous recombinant proteins. Types of the proteins include, forexample, so-called signaling related proteins, receptors, growthfactors, cell cycle related factors, transcription factors, translationfactors, transport related proteins, secretory proteins, cytoskeletalproteins, enzymes, chaperones, or disease related proteins, where thediseases include cancers, diabetes or genetic disease etc.

In the present invention, it is required to introduce a nonsense codon(an amber codon in the case of a suppressor tRNA being an ambersuppressor) or a frame shift codon into a site into which a non-naturalamino acid, in particular a lysine derivative or a tyrosine derivative,is to be incorporated, whereby a non-natural amino acid, in particular alysine derivative, can be specifically incorporated into the nonsensecodon (amber codon) site or the frame shift codon site. As used herein,a “frame shift mutation” is a mutation in an amino acid sequence thatshifts the frame to be translated that is caused by deletion orinsertion of 1, 2, or 4 bases, and the aberrant codon formed at themutated site is referred to as a “frame shift codon”. Preferably, aframe shift codon is a codon formed of 4 or 5 bases. It has been triedto extend the genetic code by using 4-base codons in various host cells.In the case of Escherichia coli, for example, the 4-base codon of AGGAis an alternative codon that is usable without causing much disturbanceof cell function (Anderson, J. C. et al., Proc. Natl. Acad. Sci., USA101, 7566-7571).

Methods for performing site-specific mutagenesis of a protein may be anywell-known methods, and are not restricted to a particular one. Forexample, such mutagenesis may be conducted as required according to amethod described in Gene 152, 271-275 (1995); Methods Enzymol. 100,468-500 (1983); Nucleic Acids Res. 12, 9441-9456 (1984); Proc. Natl.Acad. Sci. USA 82, 488-492 (1985) or “Saiboukougaku bessatsu‘Sinsaiboukougakujikken protocol’, Shyujyunshya, 241-248 (1993)”, or amethod using “QuickChange Site-Directed Mutagenesis Kit” (Stratagene)

In the present invention, expression can be performed in an animal cell,and thus a non-natural amino acid can be incorporated into such proteinthat is not or is poorly expressed in Escherichia coli or a cell-freeprotein system or that cannot undergo post-translational modificationnecessary for changing to an active form. Various types of such proteinsare known to a person of ordinary skill in the art. For example, theremay be synthesized an alloprotein of, but not restricted to, a tyrosinekinase type receptor such as human EGFR, etc. (Cell, 110, 775-787(2002)), human Groucho/TLE1 protein (Structure 10, 751-761 (2002)) orrat muscle-specific kinase (Structure 10, 1187-1196 (2002)).

In the method of the present invention, an alloprotein is expressed inan animal cell so that a non-natural amino acid, in particular a lysinederivative, can be incorporated into a carbohydrate chain-linkedglycoprotein. Particularly, in the case of a type of glycoprotein whosepattern of addition of carbohydrate chain in a cell-free protein systemis different from its original (natural) pattern, a system in an animalcell of the present invention is thought to be an effective measure toobtain an alloprotein to which is added a glycoprotein of a pattern ofinterest (an original pattern).

A protein for incorporation of a non-natural amino acid, in particular alysine derivative, may be expressed, for example, as follows: a genehaving a sequence constructed such that its codon corresponding to theposition of a desired amino acid of a desired protein is replaced with anonsense codon or a frame shift codon and a desired tag is added to theC terminus thereof is integrated into the BamHI-XhoI site of pcDNA4/TOetc. to produce a plasmid, which is introduced into an animal cell,resulting in expression thereof.

(Host)

An animal cell as host (cell) used in the present invention ispreferably a mammalian cell in which a recombinant gene system isestablished. Examples of useful mammalian host cell systems include aChinese hamster ovary (CHO) cell and a COS cell. More unique examplesinclude the SV40-transformed simian kidney CV1 system (COS-7, ATCC CRL1651); human embryo kidney system (293 cell or subcloned 293 cell forgrowth in suspension culture, J. Gen Virol., 36:59 (1977)); Chinesehamster ovary cell/-DHFR (CHO, Proc. Natl. Acad. Sci. USA, 77:4216(1980)); mouse Sertoli's cell (TM4, Biol. Reprod., 23:243-251 (1980));human lung cell (W138, ATCC CCL 75); human liver cell (Hep G2, HB 8065);and mouse breast cancer cell (MMT 060562, ATCC CCL51). An expressionsystem for each of those host cells is established, and it is within thetechnical skill of a person of ordinary skill in the art to select anappropriate host cell.

Methods for introducing a vector into the above host cells include, forexample, electroporation (Nucleic, Acids Res. 15, 1311-1326 (1987)), acalcium phosphate method (Mol. Cell Biol. 7, 2745-2752 (1987)), alipofection method (Cell 7, 1025-1037 (1994); Lamb, Nature Genetics 5,22-30 (1993)), etc. These methods may be conducted, for example, inaccord with a method described in Molecular Cloning 3^(rd) edition, ColdSpring Harbor Laboratory Press (2001), etc. In accord with one exemplaryembodiment of the present invention, there is provided a recombinanteukaryotic cell, preferably a recombinant mammalian cell, transformed ortransfected with an expression vector of the above non-eukaryoticsuppressor tRNA.

(Method for Producing a Protein with Incorporated Non-Natural AminoAcid)

As an example, expression of an alloprotein with an incorporated lysinederivative is explained below. An animal cell is incubated underappropriate conditions in a culture medium suitable for the growth ofthe animal cell (for example, Opti-MEMI (Gibco BRL), etc., in the caseof a CHO cell), the animal cell containing (A) an expression vectorexpressing an aminoacyl-tRNA synthetase, in particular a PylRS, in theanimal cell; (B) an expression vector expressing in the animal cell aMethanosarcina mazei pyrrolysine tRNA capable of binding to anon-natural amino acid, in particular a lysine derivative, in thepresence of the above aminoacyl-tRNA synthetase (in particular PylRS);(C) an expression vector expressing a desired protein subjected tononsense mutation or frame shift mutation at a desired position; and anon-natural amino acid, in particular a lysine derivative. In the caseof a CHO cell, for example, the cell is incubated at ca. 37 degreesCelsius for ca. 24 hours.

Alternatively, in the case of the above pyrrolysine tRNA expressed byusing a bacteriophage promoter, it is preferable to introduce, inaddition to the above (A) to (C), (D) a vector expressing in an animalcell an RNA polymerase gene capable of transcription of the abovebacteriophage promoter. For example, as RNA polymerases fortranscription of the above T7 promoter, T3 promoter, and SP6 promoter,there are known a T7 RNA polymerase, a T3 RNA polymerase, and SP6 RNApolymerase, respectively.

Some examples of the present invention are detailed below but it shouldbe understood that the present invention is not restricted to theexamples mentioned below.

EXAMPLES

In these examples, there were conducted experiments for incorporation ofa lysine derivative or a tyrosine derivative into position 111 of humanGrb2 and position 91 of β-galactosidase. In this regard, the Grb2 is aprotein involved in cancer by interaction with an epidermal growthfactor receptor in a cell.

(Construction of PylRS and TyrRS Expression Plasmids)

A PylRS expression plasmid was constructed by PCR amplifying a DNAsequence (SEQ ID NO:8) made up of a wild type PylRS gene ofMethanosarcina mazei, the N terminus region of which was linked to aFLAG tag. and incorporated into the NheI-BamHI site of pcDNA3.1 togenerate the expression plasmid.

On the other hand, the expression plasmid PEYSM1 of 3-iodo-L-tyrosinespecific mutant of Escherichia coli tyrosyl tRNA synthetase (TyrRSV37C195, supra., Non-Patent Document 2) has been reported. Transfectioninto a mammalian culture cell of this plasmid together with anexpression plasmid of a suppressor tRNA followed by addition of3-iodo-L-tyrosine to a cell culture medium allows incorporation of the3-iodo-L-tyrosine into the amber codon site of a protein gene with anamber mutation. The method of preparing the above expression plasmid isdescribed in the above Patent Document 1 and Non-Patent Document 2, thecontents of both documents being incorporated herein by reference. Inaddition, there has been reported mutant(s) specific to4-azido-L-phenylalanine and 4-benzoyl-L-phenylalanine (Chin et al.,supra). Those mutant TyrRSs were cloned into the multiple cloning siteof pcDNA4/TO.

(Construction of Suppressor tRNA Expression Plasmid)

A sequence (SEQ ID NO: 11) was synthesized from a DNA primer. Thissequence was made up of a wild type M. mazei pyrrolysine tRNA genelinked at the 5′ end to a human valine tRNA gene via a linker (SEQ IDNO:10) and a leader, and further linked at the 3′ end to a transcriptiontermination sequence. This sequence was introduced into pCR4Blunt-TOPO,resulting in construction of a tRNA^(VAL)-tRNA^(Pyl) tandem expressionplasmid. A tRNA^(VAL)-tRNA^(Tyr) tandem expression plasmid wasconstructed in a similar manner for expression of Escherichia colisuppressor tRNA^(Tyr).

A tRNA expression plasmid with a U6 promoter was constructed by thefollowing method. PCR was performed with a pcDNA3.1 vector template,using a primer made up of a CMV enhancer region to which a EcoRI sitewas added at the 5′ side and to which a portion of the 5′ side sequenceof a U6 promoter was added at the 3′ side. And PCR was performed with asiSTRIKE template, using a primer made up of a U6 promoter region havinga portion of the 3′ end sequence of a CMV enhancer at the 5′ side and anXbaI site added to the 3′ side. The two different PCR amplificationfragments were joined to each other by overlap PCR to produce a DNAfragment having the structure—EcoRI site/CMV enhancer/U6 promoter/XbaIsite. The so-produced fragment was treated with EcoRI and XbaI and thencloned into pUC119.

The above prepared plasmid was treated with XbaI and HindIII. Theresulting product was joined to a fragment containing atRNA^(Pyl)-terminator isolated from the previously preparedtRNA^(VAL)-tRNA^(Pyl) tandem expression plasmid by XbaI and HindIIIdigestion, as a result of which there was obtained an expression plasmidhaving a DNA fragment of a nucleotide sequence set forth in SEQ ID NO:6.A plasmid made up of pcDNA3.1+Zeo having three tandem tRNA^(Ptl) linkedto the multiple cloning site was constructed as a control.

Likewise, a similar expression plasmid, but using Escherichia colisuppressor tyrosine tRNA instead of Methanosarcina mazei wild typepyrrolysine tRNA^(Pyl), having the CMV enhancer and promoter of human U6snRNA was constructed in a similar manner (SEQ ID NO:7). Here, it hasbeen reported that the CMV enhancer activates RNA transcription from U6promoter.

(Construction of Reporter Gene Expression Plasmid)

Using a QUICK CHANGE site-directed mutagenesis kit (Stratagene), theleucine codon at position 111 of human grb2 was converted to an ambercodon (grb2 (111 amber)). Subsequently, a gene (SEQ ID NO:12)constructed such that a FLAG tag (DYKDDDDK, amino acids 217-224 in SEQID NO:12) was added to the C terminus thereof was incorporated into theBamHI-XhoI site of pcDNA4/TO to produce a plasmid for detection ofsuppression.

Likewise, a tyrosine codon at position 91 of Escherichia coliβ-galactosidase (lacZ) was converted to an amber codon (lacZ (91 amber))and cloned into the multiple cloning site of pcDNA3.1+ (Zeo resistant).

Introduction of Gene into Cell and Suppression Reaction Example 1 Grb2Amber Suppression by tRNA^(Pyl) Linked to Human Valine tRNA GenePromoter

Chinese hamster ovary cells (CHO cells) cultivated in a 2.0 ml culturescale 6-well plate (as subculture medium, DMEM/F-12 (Gibco), 10% FBS(ICN), 1/100 penicillin-streptomycin (Gibco) were used) were providedwith 0.5 μg/well of three different expression plasmids: PylRS,tRNA^(Pyl) linked to a human valine tRNA gene promoter, and grb2 (111amber) in various combinations thereof (see RESULT), and transfectionwas conducted under 90% confluent state. The transfection was performedusing Lipofectamine 2000 (Invitrogen) according to the instructionmanual from Invitrogen. Opti-MEM (Gibco) was used as the culture mediumfor the transfection. The transfected cell culture medium (solution) wasreplaced with DMEM/F-12 (Gibco) in the presence or absence of 1 mMNε-Boc-lysine (Bachem), expression of the plasmid genes was induced byaddition of 1 μg/mL tetracycline, and incubation was conducted at 37degrees Celsius for ca. 20 hours in a CO₂ incubator.

The above cultured cells from which the cell culture medium (solution)was removed were washed with buffer solution, followed by lysis of thecells to recover proteins. SDS-polyacrylamide gel electrophoresis wasperformed to separate the proteins from each other according tomolecular weights thereof, followed by electroblotting (100 V, 1 hour)to membrane. Anti-FLAG M2 (Sigma) was used as primary antibody fordetection of grb2 (111 amber) expression product, and sheep wholeantibody conjugated with horseradish peroxidase for anti-mouse IgG(Amersham) was used as secondary antibody. As a detection reagent, ECLwestern blotting detection reagent (Amersham) was used. Measurement wasconducted using a cooled CCD camera LAS 1000 plus (Fuji Film).

FIG. 2 shows a result detected by western blotting of suppression ofGrb2 (111 amb). Lane 1 on the left is a control for showing the positionof the band of full-length Grb2. Wild type Grb2 has FLAG tag added tothe C terminus thereof, and the band thereof is detected at the positionindicated by the arrow for Grb2 in cases where it is synthesized to theC terminus thereof. Lanes 2 to 4 show results in the case of lack of anyone of PylRS, pyrrolysine tRNA and Nε-Boc-lysine. In these cases, afull-length Grb2 was not synthesized. Contrary, lane shows a result inthe case of all of PylRS, pyrrolysine tRNA and Nε-Boc-lysine beingintroduced into a cell, wherein the full-length Grb2 was synthesized. Itis apparent from the results that by PylRS and pyrrolysine tRNA,Nε-Boc-lysine was incorporated into an amber codon incorporated intoposition 111 of Grb2.

Example 2 Grb2 Amber Suppression by tRNA^(Tyr) and tRNA^(Pyl) Linked toU6 Promoter

Subsequently, human Grb2 gene, wild type TyrRS, and PylRS, which wereprepared using a similar method in the above Example, were expressed andwere subjected to suppression by Methanosarcina mazei wild typepyrrolysine tRNA^(Pyl) having U6 promoter or Escherichia coli suppressortRNA^(Tyr). FIG. 3 shows a result detected by western blotting ofsuppression of Grb2 (111 amb). Lane on the most right is a control forshowing the position of bands of wild type Grb2. Two lanes on the leftshow results in cases where Escherichia coli suppressor tRNA^(Tyr)having U6 promoter was expressed. From the band of Grb2 having beendetected depending on expression of TryRS, it is apparent that tyrosinewas incorporated into the amber codon of Grb2. Two lanes in the middleshow results in cases where pyrrolysine tRNA^(Pyl) having U6 promoterwas expressed. From the band of Grb2 having been detected by addition ofNε-Boc-lysine to the culture medium, it is apparent that Nε-Boc-lysinewas incorporated into the amber codon of Grb2. The results revealed thatthe tRNA gene having a U6 promoter linked to its 5′ end was transcribedin a mammalian cell. A control experiment in which tyrosine is not addedto the culture medium in the case of tyrosine tRNA being expressed wasnot conducted because cells do not grow in the absence of tyrosine.

Example 3 Lacz Amber Suppression by tRNA^(Pyl)

Chinese hamster ovary cells (CHO-TRex cells) were seeded in a 24-wellplate at 1.2×10⁵ cells/well and incubated in DMEM/F-12 culture media(Gibco) containing 10% fetal bovine serum (ICN) and 1/100penicillin-streptomycin (Gibco). The next day, when the culture was 95%confluent, transfection was conducted by using 0.4 μg lacZ (91 amber),0.2 μg PylRS expression plasmids, and three different suppressortRNA^(Pyl) expression plasmids (each containing human valine tRNA, U6promoter, and CMV enhancer) prepared in the above Example. Thetransfection was conducted using 2 μl Lipofectamine 2000 (Invitrogen)according to the instruction manual therefor. Opti-MEM (Gibco) was usedas culture medium for the transfection.

The transducted cell culture medium was replaced with DMEM/F-12 (Gibco)in the presence or absence of 1 mM Boc-lysine (Bachem), expression wasinduced by addition of 1 μg/mL tetracycline, and incubation wasconducted at 37 degrees Celsius for ca. 20 hours in a CO₂ incubator. Thenext day, proteins were recovered from the cells, and lacZ enzymeactivities thereof were examined using a f3-Gal reporter assay kit(TOYOBO). The result is shown in FIG. 4. It is apparent therefrom thatin cases where either human valine tRNA promoter or U6 promoter wasused, β-galactosidase activity was detected by addition of Boc-lysine tothe medium, and thus the amber codon of lacZ gene was suppressed. On thecontrary, in the case of the tRNA expression vector using a CMV promoterbut not containing the human valine tRNA promoter or U6 promoter,suppression was not caused because β-galactosidase activity was notdetected regardless of whether Boc-lysine was added or not. It isassumed that expression of suppressor tRNA by U6 promoter issignificantly high as compared with that by human valine tRNA promoter.

Example 4 Lacz Amber Suppression by tRNA^(Tyr) Linked to U6 Promoter

By a method similar to that of Example 3, a U6 promoter-linkedEscherichia coli suppressor tRNA^(Tyr) gene and three different mutantTyrRS expression plasmids were expressed, and lacZ amber suppression wasperformed by addition to the culture of three types of tyrosinederivatives; iodotyrosine (IY), azidophenylalanine (AzPhe) andparabenzoylphenylalanine (pBpa). The result is shown in FIG. 5. It isapparent therefrom that in each case where any one of the amino acidswas added, significantly high β-galactosidase activity was detected ascompared with the case where no amino acids were added, and thus theamber codon of lacZ gene was suppressed. In this regard, it seems thatthe detection of β-galactosidase activity even in the absence ofiodotyrosine is due to the suppression caused even in the case of noaddition of IY by IY-specific mutant TyrRS, which incorporates not onlyiodotyrosine but also tyrosine.

Reference Example 1

FIG. 6 shows data of mass spectrometry indicating that in Escherichiacoli, Nε-Boc-lysine was incorporated into a peptide in the presence ofPylRS and pyrrolysine tRNA. As shown in FIG. 6, the peak of molecularweight (MW) 1327.67 indicates a peptide whose sequence is NSYSPILGYWK.The peak of molecular weight 1392.76 indicates a peptide whose tyrosinepresented at the 9th position from the left was replaced withNε-Boc-lysine (which is indicated with the mark “*”).

Example 5 Construction of Expression System of Suppressor tRNA by T7Promoter, and lacZ Amber Suppression

A T7 RNA polymerase gene was amplified by PCR and cloned between theEcoRI and XhoI sites of pcDNA4/TO to produce a T7 RNA polymeraseexpression plasmid. In order to produce a T7-tRNA^(Ttr) gene, first PCRwas performed using U6-tRNA^(Tyr) (SEQ ID NO:7) as template, therebyadding a T7 promoter to the tRNA^(Tyr) sequence, to be cloned intopCR4blunt-TOPO. Then, the T7-tRNA^(Tyr) gene was cut out by treatmentwith EcoRI, to be cloned into EcoRI site of pBR322. The so preparedT7-tRNA^(Tyr) gene and a portion of the plasmid sequence (SEQ ID NO:15)were amplified by PCR, and the DNA obtained was purified and used totransform cells. In order to construct a tRNA^(Pyl) expression plasmid,the pBR322 into which T7-tRNA^(Tyr) gene was cloned as mentioned abovewas treated with XbaI and HindIII, followed by isolation of a fragmentcontaining the tRNA^(Pyl)-terminator from the tRNA expression plasmidwith U6 promoter by means of XbaI and HindIII digestions, to couple themwith each other. The so prepared T7-tRNA^(Pyl) gene and a portion of aplasmid sequence (SEQ ID NO:14) were amplified by PCR, and the DNAobtained was purified and used to transform cells.

Using a method similar to that of Example 3, transfection was performedby using 0.2 μg T7-tRNA^(Pyl) expression plasmid, 0.1 μg PylRSexpression plasmid, 0.4 μg lacZ (91 amber) expression plasmid, and 0.3μg T7 RNA polymerase expression plasmid to conduct lacZ ambersuppression, except that incubation was conducted in DMEM/F-12 culturemedium (Gibco) without 1/100 penicillin-streptomycin (Gibco). The resultis shown in FIG. 7. It is apparent therefrom that in the case where T7RNA polymerase was expressed (T7RNAP+), significantly highβ-galactosidase activity was detected as compared with the case where T7RNA polymerase was not expressed (T7RNAP−), and thus the amber codon oflacZ gene was suppressed.

Using a method similar to that of Example 3, transfection was performedby using 0.18 μg T7-tRNA^(Tyr) gene DNA, 0.1 μg TyrRS expressionplasmid, 0.4 μg lacZ (91 amber) expression plasmid, and 0.3 μg T7 RNApolymerase expression plasmid to conduct lacZ amber suppression.However, incubation was conducted in DMEM/F-12 culture medium (Gibco)without 1/100 penicillin-streptomycin (Gibco). The result is shown inFIG. 8. It is apparent therefrom that in the case where T7 RNApolymerase was expressed, significantly high β-galactosidase activitywas detected as compared with the case where T7 RNA polymerase was notexpressed, and thus the amber codon of lacZ gene was suppressed.

Example 6 Construction of Expression System of Suppressor tRNA by U1snRNA-Type Transcription Promoter, and lacZ Amber Suppression

Construction of tRNA expression plasmid by U1snRNA type-promoter wasconducted according to the following method. Using the previouslyprepared U6-tRNA^(Tyr) (SEQ ID NO:7) as template, the region from 198bases upstream of the U6 promoter transcription initiation site toupstream of the TATA box was amplified by using the following primers:

(SEQ ID NO: 16) 5′-ATGATATCAGAGGGCCTATTTCCCAT-3′ (SEQ ID NO: 17)5′-TGCTCGAGAAGCCAAGAATCGAAATAC-3′.

This region includes a transcription element PSE, wherein the amplifiedDNA fragment has an EcoRV site added at its 5′ end and an XhoI siteadded at its 3′ end. The PCR product was integrated into the EcoRV-XhoIsite of plasmid pcDNA3.1+. Vector-originating EcoO109I and NotI sitesare present downstream of the XhoI site. A sequence downstream of theTATA box of the U6 promoter and a terminator for stopping transcriptionby polymerase III were inserted between the XhoI and EcoO109I sites.After insertion of the tRNA^(Tyr) sequence into the EcoO0109I site, a 3′box, which is a terminator of polymerase II, was further inserted intothe NotI site. The whole region from EcoRV through the 3′ boxconstitutes a PSE-tRNA^(Tyr) gene (SEQ ID NO:18). This gene wasamplified by PCR and cloned into pCR4blunt-TOPO to produce a plasmid,which corresponds to the PSE-tRNA^(Tyr) expression plasmid. The soprepared PSE-tRNA^(Tyr) gene has a U6 promoter from which the TATAsequence is removed so that transcription by RNA polymerase II is caused(Das et al., Nature 1995, Vol. 374, pp. 657-660). In addition, PCRamplification was performed using the following two primers having CMVenhancers similar to those of the U6 promoter:5′-ATCGAATTCTAGTTATTAATAGTAATCAATTACG-3′ (SEQ ID NO:19) and5′-AGCCTTGTATCGTATATGC-3′ (SEQ ID NO:20); and 5′ phosphorylation wasfurther conducted, followed by insertion into the EcoRI site of thePSE-tRNA^(Tyr) gene to produce CMV-DSE-PSE-tRNA^(Tyr).

Using a method similar to that of Example 3, transfection was conductedby using 0.2 μg of PSE-tRNA^(Tyr) expression plasmid orCMV-PSE-tRNA^(Tyr) expression plasmid, 0.2 μg of TyrRS expressionplasmid, and 0.4 μg of lacZ (91 amber) expression plasmid. On the dayfollowing the transfection, cells were stained using a β-GalactosidaseStaining Kit (Mirus) to examine whether amber suppression of lacZ wascaused. A cell having caused suppression is expected to be stained blue.FIG. 9 shows photographs depicting results of staining cells, whereinstained cells are indicated with arrows. Presence or absence of theenhancer did not cause a particularly large difference. The suppressionactivities, even though low, were confirmed in both cases.

INDUSTRIAL APPLICABILITY

The present invention is able to effectively produce alloprotein(s) intowhich there is incorporated a non-natural amino acid such as a lysinederivative, a tyrosine derivative, etc.

The invention claimed is:
 1. A DNA construct comprising a suppressortRNA gene derived from Escherichia coli, wherein said tRNA gene containsno internal promoter sequence that functions in a eukaryotic cell, andhas a eukaryotic promoter linked to the 5′ end of said tRNA gene whereinthe eukaryotic promoter is a pol III type III promoter.
 2. The DNAconstruct of claim 1, wherein said tRNA gene is a suppressor tyrosinetRNA gene.
 3. The DNA construct of claim 1, further comprising atranscription terminator sequence linked to the 3′ end of said tRNAgene.
 4. The DNA construct of claim 1, wherein said pol III type IIIpromoter is a promoter of a U6 snRNA gene.
 5. The DNA construct of claim4, wherein said promoter of a U6 snRNA gene has the nucleotide sequenceset forth in SEQ ID NO: 3 or a nucleotide sequence that is at least 90%identical thereto, and induces transcription by RNA polymerase III in amammalian cell.
 6. A method of synthesizing a suppressor tRNA comprisingcausing the DNA construct of claim 1 to undergo transcription in aneukaryotic cell.
 7. A recombinant eukaryotic cell that is transformed ortransfected by the DNA construct of claim
 1. 8. A process for producinga protein comprising a non-natural amino acid in a eukaryotic cellwherein the process comprises expressing in the eukaryotic cell: (a) aDNA construct that encodes a suppressor tRNA derived from Escherichiacoli, wherein said tRNA gene does not naturally contain an internalpromoter sequence that functions in a eukaryotic cell, where said tRNAis modified by linking a pol III type III eukaryotic promoter to the 5′end of said suppressor tRNA gene, (b) a polynucleotide that encodes anaminoacyl-tRNA synthetase for the non-natural amino acid, and (c) apolynucleotide encoding a desired protein that has a nonsense mutationor frame shift mutation at a desired position, and culturing saideukaryotic cell in a culture medium comprising an unnatural amino acid,thereby producing the protein comprising the unnatural amino acid. 9.The process of claim 8, wherein said tRNA gene is a suppressor tyrosinetRNA gene.
 10. The process of claim 8, wherein said non-natural aminoacid is a tyrosine derivative.
 11. The process of claim 10, wherein saidtyrosine derivative is 3-iodo-tyrosine, 4-azido-L-phenylalanine, or4-benzoyl-L-phenylalanine.
 12. The process of claim 8, wherein said polIII type III promoter is a promoter of a U6 snRNA gene.
 13. The processof claim 12, wherein said promoter of a U6 snRNA gene has the nucleotidesequence set forth in SEQ ID NO:3 or a nucleotide sequence that is atleast 90% identical thereto, and induces transcription by RNA polymeraseIII in a mammalian cell.